1. Field of the Invention
This invention relates to a novel ribozyme, a DNA fragment for the production of the ribozyme, a recombinant vector and a method for the production of the ribozyme.
2. Prior Art Statement
It had been long held that all enzymes are composed of proteins. The time-honored concept of enzymes was shattered in 1981 when a ribozyme, a RNA molecule possessing enzymatic activity, was discovered.
This epochal discovery was made by Professor T. Cech et al. of the University of Colorado, U.S.A. They demonstrated that the ribosomal RNA (rRNA) precursor of tetrahymena, a protozoan, expels the intron (IVS) unnecessary for the transfer of gene information by self-splicing without the aid of any protein ["Nature" Vol 308, pp. 820-825 (1984)].
RNA molecules which possess the function of self-splicing and ribozymes which cleave other RNA molecules have since been discovered.
Recently, J. Haseloff and W. L. Gerlach have taken an interest in a nucleotide sequence held in common among ribozymes of several species of plant viruses and have succeeded in constructing an artificial ribozyme which forms a catalytic site with only 24 bases ["Nature", Vol. 334, pp. 585-591 (1988)].
FIG. 1 illustrates the design of this artificial ribozyme. In the diagram, .sup.5' XXX . . . XXX.sup.3 represents the nucleotide sequence of a RNA molecule serving as a substrate.
This artificial ribozyme is composed of a binding site (.sub.3' YYY . . . YYY.sub.5') forming a nucleotide pair in recognition of the nucleotide sequence of the RNA molecule serving as a substrate and a catalytically active site possessing 24 specific nucleotide sequences. It effects cleavage of the RNA at the position (indicated by an arrow in the diagram) adjoining the (GUC) part of the substrate RNA.
In the aforementioned X . . . X sequence, the nucleotide sequence indicated by GUC may be changed to some other sequence.
Now, the method proposed by Haseloff et al. for the construction of this artificial ribozyme will be described. Specifically, this construction is effected by synthesizing a DNA possessing a complementary nucleotide sequence of said ribozyme RNA and coding for the ribozyme RNA on the basis of the design of ribozyme mentioned above, inserting the resultant synthetic DNA in a plasmid, transforming the resultant recombinant plasmid into a clone, cleaving the cloned DNA with a restriction enzyme Thereby obtaining a DNA fragment coding for the ribozyme RNA, and performing in vitro transcription using the DNA fragment as a template. It has been demonstrated that, in accordance with this method, three ribozymes differing in nucleotide sequence of binding site are synthesized and these three ribozymes cleave the mRNA corresponding to chloramphenicol acetyl transferase at appropriate positions.
The method described above, however, suffers from the following problems.
As described above, this method obtains the artificial ribozyme by transforming the recombinant plasmid, cleaving the resultant clone with a restriction enzyme thereby giving rise to a linearized DNA coding for the ribozyme RNA, and performing transcription using the DNA fragment as a template (run-off transcription; FIG. 2). Thus, this method inevitably entails a treatment with the restriction enzyme. The reason for this treatment of cleavage is that when the ribozyme RNA possesses an extra nucleotide sequence other than a binding site necessary for the recognition of sites of cleavage, there arises the possibility that the extra nucleotide sequence will form a inadvertent base-pairing with a nucleotide sequence other than the cleaving site of the substrate RNA and, as the result, the substrate specificity will be decreased. To prevent this phenomenon, it is necessary to ensure that surplus nucleotide sequences on the 5'-terminus site and the 3'-terminus site are not transcribed when the ribozyme is obtained using the ribozyme-coding plasmid as a template. On the 5'-terminus site, the problem may be solved as by linking the aforementioned DNA to the immediate downstream of any promoters. On the other hand, the control of the 3'-terminus site is more difficult since no universal terminator effective in terminating transcription of an inserted gene such as ribozyme has yet been discovered. The conventional method, therefore, must adopt the so-called run-off method which relies for the treatment of the 3'-terminus site of the DNA template with a restriction enzyme (FIG. 2).
The conventional method necessitates use of a cleaved linear DNA in the transcription entailing the extra work of using the restriction enzyme. While this run-off method allows in vitro synthesis of the ribozyme, it is incapable of effecting in vivo production of the ribozyme while retaining and propagating the recombinant vector including the DNA fragment which codes for the ribozyme RNA. This method experiences difficulty in cleaving and consequently rendering harmless the mRNA originating in a pathogenic virus and, at the same time, allowing the DNA coding for ribozyme to be retained in the vital animal or plant body.
An object of this invention is to provide a novel ribozyme which permits a ribozyme devoid of any surplus nucleotide sequences at its 3'- and 5'-binding sites to be obtained by transcription directly using as a template a circular DNA vector containing the ribozyme gene without necessitating cleavage with the aforementioned restriction enzyme and also permits in vivo production of the ribozyme while enabling the recombinant vector to be retained and propagated in the vital body. Another object of this invention is to provide a method for the production of the novel ribozyme.